Cardiomyocyte differentiation by gata-4 deficient es cells

In situ hybridization studies, promoter analyses, and anli sense RNA experiments have implicated transcription factor GATA-4 in the regulation of cardiomyocyte differentiation. In the present study we utilized Gata4-/- ES cells to determine whether this transcription factor is essential for cardiomyocyte lineage commitment. Wild type or Gata4-/- ES cells were differentiated into embryoid bodies using a hanging drop method. After transfer to tissue culture plates, contraction and cardiomyocyte formation were seen in 80-100% of wild type embryoid bodies, whereas only 25% of the Gata4-/- embryoid bodies contained cardiomyocytes. Indirect immunofluorescence confirmed that the cardiomyocytes in the GATA-4 deficient embryoid bodies expressed cardiac a-myosin heavy chain. To assess the capacity of Gata4-/- ES cells to differentiate in vivo, we prepared and analyzed chimeric mice. Gata4-/- ES cells were injected into morulas derived from ROSA26 mice, a transgenic line that expresses lacZ in a cell types. Chimeric embryos of varying ages were prepared and subjected to X-gal staining to discriminate ESderived tissue from morula-derived tissue. Gata4-/~ ES cells contributed to endocardium, myocardium, and epimyocardium. In situ hybridization showed that myocardium derived from Gata4-/~ cells expressed normal amounts of several cardiomyocyte-specific transcripts, including cardiac troponin C, GATA-6, and cardiac myosin light chain-2v. We conclude that GATA-4 is" not essential for cardiomyocyte differentiation, possibly because other GATA-binding proteins present in cardiac tissue (e.g. GATA-5 or GATA-6) compensate for a lack of GATA-4. Whether GATA-4 is required for full function or longevity of cardiomyocytes awaits further study, including analysis of Gata4 null mice.